Amersham Pharmacia Biotech EPS 3501 XL Manual Del Usuario página 18

EPS 3501 XL - User Manual
conductivity in the electrophoresis system, the values for the other two parameters. Voltage, current,
power and conductivity are related by the following equations:
U = I / L
P = U x I
Where U = Voltage, I = Current, P = Power and L = Conductivity
Equation (1) is more familiar if the conductivity is replaced by the reciprocal resistance (R):
U = R x I
The electric field E, measured in V/cm, is the driving force behind electrophoresis.
E = U / d
where E = Electrical field strength, U = Voltage, d = distance
The electrical field strength is achieved by applying a voltage. The higher the voltage, the faster the
electrophoresis. Fast electrophoresis is beneficial since it counteracts diffusion.
The disadvantage of increasing the voltage too much is that most of the generated electrical energy, the
product of power and time, is transformed to heat. Therefore cooling of electrophoresis equipment is
recommended. Cooling will also reduce "smiling" effects which are caused by mobility differentials
across an electrophoresis gel resulting from poor heat transfer. Since the cooling efficiency cannot be
increased indefinitely, the power should be limited when programming the power supply.
The parameter that should be chosen as the constant and thus control the electrophoresis depends on the
type of electrophoresis. In the case of homogeneous buffers throughout the system (same electrode and
gel buffer), the conductivity is constant during the electrophoresis. If the conductivity is constant, the
voltage will be proportional to the current and the power to the square of the current, according to (1)
and (2). This means that the result of the electrophoresis will be the same, regardless of which parameter
is chosen as the constant. For historical and practical reasons, voltage is most commonly used for
regulation. Submarine gel electrophoresis of DNA/RNA and pulsed field electrophoresis are usually run
at constant voltage. SDS-PAGE using continuous buffer systems is run at constant voltage or current.
For discontinuous buffer systems, the resistance will increase as the electrophoresis proceeds due to a
decrease in conductivity. Running at constant voltage will result in decreasing current and power.
Constant voltage will thus be "safe" in the respect that the power will not increase and produce more
and more heat. On the other hand, the separation will slow down and impair resolution due to an
increased time available for diffusion. Running at constant power would give a faster electrophoresis and
controlled power, while running at constant current would, at the first sight, seem to be problematic
because of increasing voltage and power.
During discontinuous electrophoresis, however, the voltage is not distributed evenly across the gel. These
gels have a region with low ionic strength that causes a high electrical field strength. This region
increases as the electrophoresis proceeds. This means that the main part of the voltage is spread over a
greater and greater distance and a higher and higher power is tolerated. This is the reason why constant
current is chosen for such applications. It is, however, recommended to also limit the power as a
precaution against overheating the gel. The power supply will probably switch over to limiting power at
the end of the run due to increased voltage.
The crossing-over between different parameters controlling the electrophoresis can be illustrated by IEF
(isoelectric focusing) using carrier ampholytes. A graphical representation of the changes in power,
voltage and current that may occur during a typical IEF run is given in Fig. 9. Since the pK values of the
carrier ampholytes and the proteins are temperature dependent, IEF must be carried out at a constant
temperature. Therefore cooling of electrophoresis equipment and controlling by power is recommended.
The main part of the IEF is thus controlled by power (phase II). The conductivity is gradually decreasing
because the carrier ampholytes and sample will loose their net charge during the build up of the pH
gradient. Thus the current will decrease and the voltage increase at constant power. During the early
stage of the formation of the pH gradient it is important to limit the current. Otherwise the gradient will
be irregularly shaped (phase I). The conductivity is not constant along the gel so it is important to also
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(1)
(2)
(Ohm´s law)